Use of titanium nitride as a counter electrode

ABSTRACT

A nanopore cell includes a titanium nitride (TiN) counter electrode configured to be at a first electric potential. The nanopore cell also include a working electrode configured to be at a second electric potential and an insulating wall. The insulating wall and the working electrode form at least a portion of a well configured to contain an electrolyte at a voltage that is at least a portion of an electric potential difference between the first electric potential of the titanium nitride counter electrode and the second electric potential of the working electrode.

CROSS-REFERENCE TO OTHER APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/286,826 entitled USE OF TITANIUM NITRIDE AS AN INTERMEDIATE LAYERIN BONDING GLASS TO A SILICON CHIP filed Jan. 25, 2016, and claimspriority to U.S. Provisional Patent Application No. 62/281,662 entitledUSE OF TITANIUM NITRIDE AS AN INTERMEDIATE LAYER IN BONDING GLASS TO ASILICON CHIP filed Jan. 21, 2016, both of which are incorporated hereinby reference for all purposes.

BACKGROUND OF THE INVENTION

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. It would be desirable to develop techniques forbiochips that make them more robust, efficient, and cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore-basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore-based sequencing chip.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore-based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state.

FIG. 7A illustrates an additional embodiment of a circuitry 700 in acell of a nanopore-based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 7B illustrates an additional embodiment of a circuitry 701 in acell of a nanopore-based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 7C illustrates a double layer that is formed at any interfacebetween a conductive electrode and an adjacent liquid electrolyte. Inthe example shown, the electrode surface is negatively charged,resulting in the accumulation of positively charged species in theelectrolyte. In another example, the polarity of all charges shown maybe opposite to the example shown.

FIG. 7D illustrates a pseudocapacitance effect that can be formed,simultaneously with the formation of a double-layer as in FIG. 7C, at aninterface between a conductive electrode and an adjacent liquidelectrolyte.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane.

FIG. 9 illustrates an embodiment of a plot of the voltage applied acrossthe nanopore versus time when process 800 is performed and repeatedthree times.

FIG. 10 illustrates an embodiment of the plots of the voltage appliedacross the nanopore versus time when the nanopore is in differentstates.

FIG. 11 illustrates an embodiment of a non-faradaic electrochemical cell1100 of a nanopore-based sequencing chip that includes a TiN workingelectrode with increased electrochemical capacitance.

FIG. 12 illustrates a top view of a plurality of circular openings 1202of a plurality of wells in a nanopore-based sequencing chip.

FIG. 13 illustrates an embodiment of a process for constructing anon-faradaic electrochemical cell of a nanopore-based sequencing chipthat includes a TiN working electrode with increased electrochemicalcapacitance.

FIG. 14 illustrates a cross-section view of a spongy and porous TiNlayer 1402 deposited above a metal layer 1404.

FIG. 15 illustrates another cross-section view of a spongy and porousTiN layer 1502 with TiN columnar structures that are grown from thesurfaces of the hole.

FIG. 16 and FIG. 17 illustrate a nanopore-based sequencing chip with oneor more “dead” zones in the flow chamber.

FIG. 18 is a diagram illustrating an embodiment of a nanopore-basedsequencing system with a serpentine flow channel.

FIG. 19 illustrates an exemplary view of an embodiment of ananopore-based sequencing chip 1904 and a flow channel component 1902that can be bonded together.

FIG. 20 illustrates two exemplary views of one embodiment of ananopore-based sequencing chip 2004 and a flow channel component 2002that can be bonded together.

FIG. 21 illustrates an exemplary view of one embodiment of ananopore-based sequencing chip 2104 and a flow channel component 2102that can be bonded together.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions across the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore-based sequencing chip may be used for DNA sequencing. Ananopore-based sequencing chip incorporates a large number of sensorcells configured as an array. For example, an array of one million cellsmay include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore-basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing protein nanopore transmembrane molecularcomplexes (PNTMC) and the analyte of interest is placed directly ontothe surface of the cell. A single PNTMC 104 is inserted into membrane102 by electroporation. The individual membranes in the array areneither chemically nor electrically connected to each other. Thus, eachcell in the array is an independent sequencing machine, producing dataunique to the single polymer molecule associated with the PNTMC. PNTMC104 operates on the analytes and modulates the ionic current through theotherwise impermeable bilayer.

With continued reference to FIG. 1, analog measurement circuitry 112 isconnected to an electrode 110 covered by a thin film of electrolyte 108.The thin film of electrolyte 108 is isolated from the bulk electrolyte114 by the ion-impermeable membrane 102. PNTMC 104 crosses membrane 102and provides the only path for ionic current to flow from the bulkliquid to working electrode 110. The cell also includes a counterelectrode (CE) 116. The cell also includes a reference electrode 117,which acts as an electrochemical potential sensor.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. At stage A, a tagged nucleotide (one offour different types: A, T, G, or C) is not associated with thepolymerase. At stage B, a tagged nucleotide is associated with thepolymerase. At stage C, the polymerase is in close proximity to thenanopore. The tag is pulled into the nanopore by an electrical fieldgenerated by a voltage applied across the membrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage B.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜300 pico Siemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS corresponding toone of the four types of tagged nucleotides. The polymerase undergoes anisomerization and a transphosphorylation reaction to incorporate thenucleotide into the growing nucleic acid molecule and release the tagmolecule. In particular, as the tag is held in the nanopore, a uniqueconductance signal (e.g., see signal 210 in FIG. 2) is generated due tothe tag's distinct chemical structures, thereby identifying the addedbase electronically. Repeating the cycle (i.e., stage A through E orstage A through F) allows for the sequencing of the nucleic acidmolecule. At stage D, the released tag passes through the nanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4. The unincorporated nucleotide can be detectedby the nanopore in some instances, but the method provides a means fordistinguishing between an incorporated nucleotide and an unincorporatednucleotide based at least in part on the time for which the nucleotideis detected in the nanopore. Tags bound to unincorporated nucleotidespass through the nanopore quickly and are detected for a short period oftime (e.g., less than 10 ms), while tags bound to incorporatednucleotides are loaded into the nanopore and detected for a long periodof time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore-based sequencing chip. As mentioned above, when the tag is heldin nanopore 502, a unique conductance signal (e.g., see signal 210 inFIG. 2) is generated due to the tag's distinct chemical structures,thereby identifying the added base electronically. The circuitry in FIG.5 maintains a constant voltage across nanopore 502 when the current flowis measured. In particular, the circuitry includes an operationalamplifier 504 and a pass device 506 that maintain a constant voltageequal to V_(a) or V_(b) across nanopore 502. The current flowing throughnanopore 502 is integrated at a capacitor n_(cap) 508 and measured by anAnalog-to-Digital (ADC) converter 510.

However, circuitry 500 has a number of drawbacks. One of the drawbacksis that circuitry 500 only measures unidirectional current flow. Anotherdrawback is that operational amplifier 504 in circuitry 500 mayintroduce a number of performance issues. For example, the offsetvoltage and the temperature drift of operational amplifier 504 may causethe actual voltage applied across nanopore 502 to vary across differentcells. The actual voltage applied across nanopore 502 may drift by tensof millivolts above or below the desired value, thereby causingsignificant measurement inaccuracies. In addition, the operationalamplifier noise may cause additional detection errors. Another drawbackis that the portions of the circuitry for maintaining a constant voltageacross the nanopore while current flow measurements are made arearea-intensive. For example, operational amplifier 504 occupiessignificantly more space in a cell than other components. As thenanopore-based sequencing chip is scaled to include more and more cells,the area occupied by the operational amplifiers may increase to anunattainable size. Unfortunately, shrinking the operational amplifier'ssize in a nanopore-based sequencing chip with a large-sized array mayraise other performance issues. For example, it may exacerbate theoffset and noise problems in the cells even further.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore-based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. One of the possible statesof the nanopore is an open-channel state when a tag-attachedpolyphosphate is absent from the barrel of the nanopore. Another fourpossible states of the nanopore correspond to the states when the fourdifferent types of tag-attached polyphosphate (A, T, G, or C) are heldin the barrel of the nanopore. Yet another possible state of thenanopore is when the membrane is ruptured. FIGS. 7A and 7B illustrateadditional embodiments of a circuitry (700 and 701) in a cell of ananopore-based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. In the above circuits, theoperational amplifier is no longer required.

FIG. 6 shows a nanopore 602 that is inserted into a membrane 612, andnanopore 602 and membrane 612 are situated between a cell workingelectrode 614 and a counter electrode 616, such that a voltage isapplied across nanopore 602. Nanopore 602 is also in contact with a bulkliquid/electrolyte 618. Note that nanopore 602 and membrane 612 aredrawn upside down as compared to the nanopore and membrane in FIG. 1.Hereinafter, a cell is meant to include at least a membrane, a nanopore,a working cell electrode, and the associated circuitry. In someembodiments, the counter electrode is shared between a plurality ofcells, and is therefore also referred to as a common electrode. Thecommon electrode can be configured to apply a common potential to thebulk liquid in contact with the nanopores in the measurement cells. Thecommon potential and the common electrode are common to (e.g., sharedamong) all of the measurement cells. There is a working cell electrodewithin each measurement cell; in contrast to the common electrode,working cell electrode 614 is configurable to apply a distinct potentialthat is independent from the working cell electrodes in othermeasurement cells.

In FIGS. 7A and 7B, instead of showing a nanopore inserted in a membraneand the liquid surrounding the nanopore, an electrical model 702representing the electrical properties of the nanopore and the membraneand an electrical model 714 representing the electrical properties ofthe working electrode are shown. Note in FIGS. 7A and 7B that therespective circuitry does not require an extra capacitor (e.g., n_(cap)508 in FIG. 5) to be fabricated on-chip, thereby facilitating thereduction in size of the nanopore-based sequencing chip.

Electrical model 702 includes a capacitor 706 that models a capacitanceassociated with the membrane (C_(membrane)) and a resistor 704 thatmodels a resistance associated with the nanopore in different states(e.g., the open-channel state or the states corresponding to havingdifferent types of tags or molecules inside the nanopore). Electricalmodel 714 includes a capacitor 716 that models a capacitance associatedwith the working electrode. The capacitance associated with the workingelectrode is also referred to as an electrochemical capacitance(C_(electrochemical)). The electrochemical capacitanceC_(electrochemical) associated with the working electrode includes adouble-layer capacitance and may further include a pseudocapacitance.

FIG. 7C illustrates a double layer that is formed at any interfacebetween a conductive electrode and an adjacent liquid electrolyte. If avoltage is applied, electronic charges (positive or negative) accumulatein the electrode at the interface between the conductive electrode andadjacent liquid electrolyte. The charge in the electrode is balanced byreorientation of dipoles and accumulation of ions of opposite charge inthe electrolyte near the interface. The accumulation of charges oneither side of the interface between electrode and electrolyte,separated by a small distance due to the finite size of charged speciesand solvent molecules in the electrolyte, acts like a dielectric in aconventional capacitor. The term “double layer” refers to the ensembleof electronic and ionic charge distribution in the vicinity of theinterface between the electrode and electrolyte.

FIG. 7D illustrates a pseudocapacitance effect that can be formed,simultaneously with the formation of a double-layer as in FIG. 7C, at aninterface between a conductive electrode and an adjacent liquidelectrolyte. A pseudocapacitor stores electrical energy faradaically byelectron charge transfer between the electrode and the electrolyte. Thisis accomplished through electrosorption, reduction-oxidation reactions,or intercalation processes.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane. Process 800 may be performed using the circuitries shown inFIG. 6, 7A, or 7B. FIG. 9 illustrates an embodiment of a plot of thevoltage applied across the nanopore versus time when process 800 isperformed and repeated three times. The voltage across the nanoporechanges over time. The rate of the voltage decay (i.e., the steepness ofthe slope of the voltage across the nanopore versus time plot) dependson the cell resistance (e.g., the resistance of resistor 704 in FIG.7A). More particularly, as the resistances associated with the nanoporein different states (e.g., the states corresponding to having differenttypes of molecules inside the nanopore) are different due to themolecules' distinct chemical structure, different corresponding rates ofvoltage decay may be observed and thus may be used to identify themolecule in the nanopore.

FIG. 10 illustrates the plots of the voltage applied across the nanoporeversus time when the nanopore is in different states. Curve 1002 showsthe rate of voltage decay during an open-channel state. In someembodiments, the resistance associated with the nanopore in anopen-channel state is in the range of 100 Mohm to 20 Gohm. Curves 1004,1006, 1008, and 1010 show the different rates of voltage decaycorresponding to the four capture states when the four different typesof tag-attached polyphosphate (A, T, G, or C) are held in the barrel ofthe nanopore. In some embodiments, the resistance associated with thenanopore in a capture state is within the range of 200 Mohm to 40 Gohm.Note that the slope of each of the plots is distinguishable from eachother.

Allowing the voltage applied across the nanopore to decay over a timeperiod during which the nanopore is in a particular detectable state hasmany advantages. One of the advantages is that the elimination of theoperational amplifier, the pass device, and the capacitor (e.g., n_(cap)508 in FIG. 5) that are otherwise fabricated on-chip in the cellcircuitry significantly reduces the footprint of a single cell in thenanopore-based sequencing chip, thereby facilitating the scaling of thenanopore-based sequencing chip to include more and more cells (e.g.,incorporating millions of cells in a nanopore-based sequencing chip).The capacitance in parallel with the nanopore includes two portions: thecapacitance associated with the membrane and the capacitance associatedwith the integrated chip (IC). Due to the thin nature of the membrane,the capacitance associated with the membrane alone can suffice to createthe required RC time constant without the need for additional on-chipcapacitance, thereby allowing significant reduction in cell size andchip size.

Another advantage is that the circuitry of a cell does not suffer fromoffset inaccuracies because V_(prc) is applied directly to the workingelectrode without any intervening circuitry. Another advantage is thatsince no switches are being opened or closed during the measurementintervals, the amount of charge injection is minimized.

Furthermore, the technique described above operates equally well usingpositive voltages or negative voltages. Bidirectional measurements havebeen shown to be helpful in characterizing a molecular complex. Forexample, they can be used to correct for baseline drift arising fromAC-non-faradaic operation.

Increased cell performance of the nanopore-based sequencing chip may beachieved by maximizing the electrochemical capacitance (seeC_(electrochemical) 716 of FIGS. 7A and 7B) associated with the workingelectrode. By maximizing C_(electrochemical), the information signalmeasured by the circuitries shown in FIG. 6, 7A, or 7B becomes morestable and the spurious signal convoluted on top of the informationsignal is minimized. C_(electrochemical) is maximized such that theimpedance associated with C_(electrochemical) is close to an AC(alternating current) short circuit compared with the impedanceassociated with C_(membrane) (see C_(membrane) 706 of FIGS. 7A and 7B).

In the present application, a non-faradaic electrochemical cell fornucleic acid sequencing that includes a titanium nitride (TiN) workingelectrode with increased electrochemical capacitance is disclosed. Aswill be described in greater detail below, the TiN working electrode isgrown and deposited in such a manner that a rough, spongy, and porouselectrode with sparsely-spaced columnar structures of TiN is formed.

FIG. 11 illustrates an embodiment of a non-faradaic electrochemical cell1100 of a nanopore-based sequencing chip that includes a TiN workingelectrode with increased electrochemical capacitance. Cell 1100 includesa conductive or metal layer 1101. Metal layer 1101 connects cell 1100 tothe remaining portions of the nanopore-based sequencing chip. In someembodiments, metal layer 1101 is the metal 6 layer (M6). Cell 1100further includes a working electrode 1102 and a dielectric layer 1103above metal layer 1101. In some embodiments, working electrode 1102 iscircular or octagonal in shape and dielectric layer 1103 forms the wallssurrounding working electrode 1102. Cell 1100 further includes adielectric layer 1104 above working electrode 1102 and dielectric layer1103. Dielectric layer 1104 forms the insulating walls surrounding awell 1105. In some embodiments, dielectric layer 1103 and dielectriclayer 1104 together form a single piece of dielectric. Dielectric layer1103 is the portion that is disposed horizontally adjacent to workingelectrode 1102, and dielectric layer 1104 is the portion that isdisposed above and covering a portion of the working electrode. In someembodiments, dielectric layer 1103 and dielectric layer 1104 areseparate pieces of dielectric and they may be grown separately. Well1105 has an opening above an uncovered portion of the working electrode.In some embodiments, the opening above the uncovered portion of theworking electrode is circular or octogonal in shape. FIG. 12 illustratesa top view of a plurality of circular openings 1202 of a plurality ofwells in a nanopore-based sequencing chip.

Inside well 1105, a film of salt solution/electrolyte 1106 is depositedabove working electrode 1102. Salt solution 1106 may include one of thefollowing: lithium chloride (LiCl), sodium chloride (NaCl), potassiumchloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂). In some embodiments, the filmof salt solution 1106 has a thickness of about three microns (μm). Thethickness of the film of salt solution 1106 may range from 0-5 microns.

Dielectric material used to form dielectric layers 1103 and 1104includes glass, oxide, silicon mononitride (SiN), and the like. The topsurface of dielectric layer 1104 may be silanized. Silanization forms ahydrophobic layer 1120 above the top surface of dielectric layer 1104.In some embodiments, hydrophobic layer 1120 has a thickness of about 1.5nanometer (nm). Alternatively, dielectric material that is hydrophobicsuch as hafnium oxide may be used to form dielectric layer 1104.

As shown in FIG. 11, a membrane is formed on top of dielectric layer1104 and spans across well 1105. For example, the membrane includes alipid monolayer 1118 formed on top of hydrophobic layer 1120 and as themembrane reaches the opening of well 1105, the lipid monolayertransitions to a lipid bilayer 1114 that spans across the opening of thewell. Hydrophobic layer 1120 facilitates the formation of lipidmonolayer 1118 above dielectric layer 1104 and the transition from alipid monolayer to a lipid bilayer. A bulk electrolyte 1108 containingprotein nanopore transmembrane molecular complexes (PNTMC) and theanalyte of interest is placed directly above the well. A singlePNTMC/nanopore 1116 is inserted into lipid bilayer 1114 byelectroporation. Nanopore 1116 crosses lipid bilayer 1114 and providesthe only path for ionic flow from bulk electrolyte 1108 to workingelectrode 1102. Bulk electrolyte 1108 may further include one of thefollowing: lithium chloride (LiCl), sodium chloride (NaCl), potassiumchloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂).

Working electrode 1102 is a titanium nitride (TiN) working electrodewith increased electrochemical capacitance. The electrochemicalcapacitance associated with working electrode 1102 may be increased bymaximizing the specific surface area of the electrode. The specificsurface area of working electrode 1102 is the total surface area of theelectrode per unit of mass (e.g., m²/kg) or per unit of volume (e.g.,m²/m³ or m⁻¹) or per unit of base area (e.g., m²/m²) As the surface areaincreases, the electrochemical capacitance of the working electrodeincreases, and a greater amount of ions can be displaced with the sameapplied potential before the capacitor becomes charged. The surface areaof working electrode 1102 may be increased by making the TiN electrode“spongy” or porous. The TiN sponge soaks up electrolyte and creates alarge effective surface area in contact with the electrolyte.

Cell 1100 includes a counter electrode (CE) 1110. Cell 1100 alsoincludes a reference electrode 1112, which acts as an electrochemicalpotential sensor. In some embodiments, counter electrode 1110 is sharedbetween a plurality of cells, and is therefore also referred to as acommon electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells. In some embodiments, counterelectrode 1110 is at least in part made of titanium nitride (TiN). Forexample, TiN has been sputtered on to a base material (e.g., glass,stainless steel, metal, etc.) to form counter electrode 1110. Using TiNto form the counter electrode has a number of advantages. TiN may beutilized due to its beneficial electrochemical properties, its generalavailability in and compatibility with existing standard semiconductormanufacturing processes, its compatibility with the biological andchemical reagents, and its relative low cost.

The ratio of the capacitance associated with the membrane (seeC_(membrane) 706 of FIGS. 7A and 7B) and the capacitance associated withthe working electrode (see C_(electrochemical) 716 of FIGS. 7A and 7B)may be adjusted to achieve optimal overall system performance. Increasedsystem performance may be achieved by reducing C_(membrane) whilemaximizing C_(electrochemical). C_(membrane) is adjusted to create therequired RC time constant without the need for additional on-chipcapacitance, thereby allowing a significant reduction in cell size andchip size.

In cell 1100, the base surface area of the opening of well 1105 (whichis the same as the base surface area of lipid bilayer 1114) and the basesurface area of working electrode 1102 are determined by the dimensionsof dielectric layer 1104 and dielectric layer 1103, respectively. Thebase surface area of working electrode 1102 is greater than or equal tothe base surface area of the opening of well 1105. Therefore, the twobase surface areas may be optimized independently to provide the desiredratio between C_(membrane) and C_(electrochemical). As shown in FIG. 11,a portion of working electrode 1102 is covered by dielectric 1104 andtherefore the covered portion does not have direct contact with saltsolution/electrolyte 1106. By using a spongy and porous TiN workingelectrode, the electrolyte can diffuse through the spaces between thecolumnar TiN structures and vertically down the uncovered portion of theworking electrode and then horizontally to the covered portion ofworking electrode 1102 that is underneath dielectric layer 1104. As aresult, the effective surface area of TiN that is in contact with theelectrolyte is maximized and C_(electrochemical) is maximized.

FIG. 13 illustrates an embodiment of a process for constructing anon-faradaic electrochemical cell of a nanopore-based sequencing chipthat includes a TiN working electrode with increased electrochemicalcapacitance.

At step A, a layer of dielectric 1304 (e.g., SiO₂) is disposed on top ofa conductive layer 1302 (e.g, M6). The conductive layer includescircuitries that deliver the signals from the cell to the rest of thechip. For example, the circuitries deliver signals from the cell to anintegrating capacitor. In some embodiments, the layer of dielectric 1304has a thickness of about 400 nm.

At step B, the layer of dielectric 1304 is etched to create a hole 1306.The hole 1306 provides a space for growing the spongy and porous TiNelectrode.

At step C, a spongy and porous TiN layer 1308 is deposited to fill thehole 1306 created at step B. The spongy and porous TiN layer 1308 isgrown and deposited in a manner to create rough, sparsely-spaced TiNcolumnar structures or columns of TiN crystals that provide a highspecific surface area that can come in contact with an electrolyte. Thelayer of spongy and porous TiN layer 1308 can be deposited usingdifferent deposition techniques, including atomic layer deposition,chemical vapor deposition, physical vapor deposition (PVD) sputteringdeposition, and the like. For example, layer 1308 may be deposited bychemical vapor deposition using TiCl₄ in combination with nitrogencontaining precursors (e.g., NH₃ or N₂). Layer 1308 may also bedeposited by chemical vapor deposition using TiCl₄ in combination withtitanium and nitrogen containing precursors (e.g.,tetrakis-(dimethylamido) titanium (TDMAT) or tetrakis-(diethylamido)titanium TDEAT). Layer 1308 may also be deposited by PVD sputteringdeposition. For example, titanium can be reactively sputtered in an N₂environment or directly sputtered from a TiN target. The conditions ofeach of the deposition methods may be tuned in such a way to depositsparsely-spaced TiN columnar structures or columns of TiN crystals. Forexample, when layer 1308 is deposited by DC (direct current) reactivemagnetron sputtering from a titanium (Ti) target, the deposition systemcan be tuned to use a low temperature, low substrate bias voltage (theDC voltage between the silicon substrate and the Ti target), and highpressure (e.g., 25 mT) such that the TiN can be deposited more slowlyand more gently to form columns of TiN crystals. In some embodiments,the depth of the deposited layer 1308 is about 1.5 times the depth ofhole 1306. The depth of the deposited layer 1308 is between 500angstroms to 3 microns thick. The diameter or width of the depositedlayer 1308 is between 20 nm to 100 microns.

FIG. 14 illustrates a cross-section view of a spongy and porous TiNlayer 1402 deposited above a metal layer 1404. As shown in FIG. 14, thespongy and porous TiN layer 1402 includes grass-like columnarstructures. FIG. 15 illustrates another cross-section view of a spongyand porous TiN layer 1502 with TiN columnar structures that are grownfrom the surfaces of the hole.

With continued reference to FIG. 13, at step D, the excess TiN layer isremoved. For example, the excess TiN layer may be removed using chemicalmechanical polishing (CMP) techniques. The remaining TiN deposited inthe hole 1306 forms a spongy and porous TiN working electrode 1310.After working electrode 1310 is formed, a layer of dielectric 1312 (e.g,SiO₂) is deposited on top of the dielectric 1304 and working electrode1310. In some embodiments, the depth of dielectric 1312 is between 100nm to 5 microns.

At step E, the layer of dielectric 1312 is etched to create a well 1314exposing only a portion of the upper base surface area of the workingelectrode. For example, the well may be etched by reactive-ion etching(RIE). Because the base surface area (e.g., π×(d1/2)²) of the opening ofthe well is independent from the base surface area (e.g., π×(d2/2)²)ofthe working electrode, C_(membrane) and C_(electrochemical) in the cellmay be fine tuned to obtain the desired C_(membrane) andC_(electrochemical) ratio. In some embodiments, the diameter (d2) ofwell 1314 is between 20 nm to 100 microns.

Building a non-faradaic electrochemical cell 1100 of a nanopore-basedsequencing chip with a spongy TiN working electrode has many advantages.Depending on the thickness of the TiN electrode (e.g., 500 angstroms to3 microns thick), the specific surface area of the spongy TiN workingelectrode and its electrochemical capacitance (e.g., 5 picofarads to 500picofarads per square micron of base area) have a 10-1000 timesimprovement over that of a flat TiN working electrode with substantiallyidentical dimensions (e.g., substantially identical thickness and basesurface area). Since the spongy TiN working electrode allows electrolyteto diffuse through easily, the diameter/width of the spongy TiN workingelectrode may extend beyond the diameter/width of the well, such thatthe base surface area of the well and the working electrode can beoptimized independently to provide the desired ratio betweenC_(membrane) and C_(electrochemical) for improved system performance.Other advantages of using TiN include its low cost and ease ofpatterning and etching compared to other electrode materials, such asplatinum.

FIGS. 16 and 17 illustrate exemplary flows of fluids across ananopore-based sequencing chip. In some embodiments, multiple fluidswith significantly different properties (e.g., compressibility,hydrophobicity, and viscosity) are flowed over an array of sensors onthe surface of the nanopore-based sequencing chip. For improvedefficiency, each of the sensors in the array should be exposed to thefluids or gases in a consistent manner. For example, each of thedifferent types of fluids should be flowed over the nanopore-basedsequencing chip such that the fluid or gas may be delivered to the chip,evenly coating and contacting each of the cells' surface, and thendelivered out of the chip. As described above, a nanopore-basedsequencing chip incorporates a large number of sensor cells configuredas an array. As the nanopore-based sequencing chip is scaled to includemore and more cells, achieving an even flow of the different types offluids or gases across the cells of the chip becomes more challenging.

In FIG. 16, an inlet (e.g., a tube) 1604 delivers a fluid to ananopore-based sequencing chip 1602, and an outlet 1606 delivers thefluid or gas out of the chip. Due to the difference in width between theinlet and the nanopore-based sequencing chip, as the fluid or gas enterschip 1602, the fluid or gas flows through paths that cover the cellsthat are close to the outer perimeter but not the cells in the centerportion of the chip.

In FIG. 17, an inlet 1710 delivers a fluid to a nanopore-basedsequencing chip 1708, and an outlet 1712 delivers the fluid or gas outof the chip. As the fluid or gas enters chip 1708, the fluid or gasflows through paths that cover the cells that are close to the centerportion of the chip but not the cells that are close to the outerperimeter of the chip.

As shown in FIG. 16 and FIG. 17, the nanopore-based sequencing chip hasone or more “dead” zones in the flow chamber. In the embodiment shown inFIG. 16, the dead zones are distributed close to the center of the chip.In the embodiment shown in FIG. 17, the dead zones are distributed closeto the outer perimeter of the chip. The sensors in the chip arraybeneath the dead zones are exposed to a small amount of the fluid or aslow flow of the fluid, while the sensors outside of the dead zones areexposed to an excess or fast flow of the fluid.

FIG. 18 is a diagram illustrating an embodiment of a nanopore-basedsequencing system with a serpentine flow channel. In some embodiments, ananopore-based sequencing system includes an improved flow chamberhaving a serpentine fluid flow channel that directs the fluids totraverse over different sensors of the chip along the length of thechannel. FIG. 18 illustrates the top view of a nanopore-based sequencingsystem 1800 with an improved flow chamber enclosing a silicon chip thatallows liquids and gases to pass over and contact sensors on the chipsurface. The flow chamber includes a serpentine or winding flow channel1808 that directs the fluids to flow directly above a single column (ora single row) of sensor banks 1806 from one end of the chip to theopposite end and then directs the fluids to repeatedly loop back andflow directly above other adjacent columns of sensor banks until all ofthe sensor banks have been traversed at least once. As shown in FIG. 18,system 1800 includes an inlet 1802 and an outlet 1804 and the serpentineor winding flow channel 1808 directs a fluid to flow from the inlet 1802to the outlet 1804 over all 16 sensor banks 1806.

With reference to FIG. 18, a fluid is directed into system 1800 throughinlet 1802. Inlet 1802 may be a tube or a needle. For example, the tubeor needle may have a diameter of one millimeter. Instead of feeding thefluid or gas directly into a wide flow chamber with a single continuousspace, inlet 1802 feeds the fluid or gas into a serpentine flow channel1808 that directs the fluid or gas to flow directly above the columns ofsensor banks 1806 serially connected together through serpentine flowchannel 1808. The serpentine channel 1808 may be formed by stackingtogether a top plate and a gasket with dividers 1810 that divide thechamber into the serpentine channel to form a flow channel component,and then mounting the flow channel component on top of the chip. Oncethe fluid or gas flows through the serpentine channel 1808, the fluid orgas is directed up through outlet 1804 and out of system 1800.

System 1800 allows the fluids to flow more evenly on top of all thesensors on the chip surface. The channel width is configured to benarrow enough such that capillary action has an effect. Moreparticularly, the surface tension (which is caused by cohesion withinthe fluid) and adhesive forces between the fluid and the enclosingsurfaces act to hold the fluid together, thereby preventing the fluid orthe air bubbles from breaking up and creating dead zones. For example,the channel may have a width of 1 millimeter or less. The narrow channelenables controlled flow of the fluids and minimizes the amount ofremnants from a previous flow of fluids or gases.

In some embodiments, the flow channel component may be bonded on top ofthe chip using room temperature bonding. Room temperature bonding joinstwo materials by heating an intermediate metal layer. The two materialsmay be similar materials or dissimilar materials. The intermediate metallayer is bombarded by a laser that creates a local heat zone. The hotzone fuses the two materials together.

FIG. 19 illustrates an exemplary view of an embodiment of ananopore-based sequencing chip 1904 and a flow channel component 1902that can be bonded together. Nanopore-based sequencing chip 1904 andflow channel component 1902 may be included in system 1800 of FIG. 18.In some embodiments, flow channel component 1902 is formed by stackingtogether a top plate and a gasket with dividers that divide the chamberinto the serpentine channel. A counter electrode 1906 is located on thebottom side of the top plate. The counter electrode's serpentine shapematches with the serpentine channel of the gasket, such that the counterelectrode is positioned directly above the sensor banks without beingblocked by the dividers of the gasket. The dividers are disposed betweenthe sensor banks so that the dividers do not block the flow of thefluids or gases over the sensor banks. In some embodiments, flow channelcomponent 1902 is a single unit formed using a glass material. Counterelectrode 1906 (e.g., top surface inside the shown serpentine channel)is formed by sputtering or coating a base material (e.g., stainlesssteel, metal other than titanium nitride, etc.) with titanium nitride(TiN). Using TiN to form the counter electrode may have a number ofadvantages. In some embodiments, TiN is a preferred material because ofits certain beneficial electrochemical properties, its generalavailability in and compatibility with existing standard semiconductormanufacturing processes, its compatibility with the biological andchemical reagents, and its relatively low cost.

Additional materials deposited in the flow channel component canpotentially react with the reagents, which is generally problematicbecause it may impose new limitations, including biocompatibilitylimitations. It is therefore preferable to limit the number of materialsused in forming the flow channel component. Therefore, it may bebeneficial to also use TiN for the aforementioned fusing process toattach the flow channel component to the die/chip.

FIG. 20 illustrates two exemplary views of one embodiment of ananopore-based sequencing chip 2004 and a flow channel component 2002that can be bonded together. Sequencing chip 2004 and flow channelcomponent 2002 may be included in system 1800 of FIG. 18. A counterelectrode 2006 is located on the top surface in the channel of flowchannel component 2002. Counter electrode 2006 (e.g., at least topsurface of inside the shown serpentine channel) is formed by sputteringtitanium nitride (TiN). The TiN metal for fusing nanopore-basedsequencing chip 2004 and flow channel component 2002 together isdeposited on the surface 2008 (e.g., shown shaded surface) of sequencingchip 2004. This may have certain advantages in allowing for a wide rangeof laser power levels to achieve the fusing without destroying circuitryin the semiconductor die beneath.

FIG. 21 illustrates an exemplary view of one embodiment of ananopore-based sequencing chip 2104 and a flow channel component 2102that can be bonded together. Sequencing chip 2104 and flow channelcomponent 2102 may be included in system 1800 of FIG. 18. TiN issputtered onto the bottom side 2106 of the flow channel component 2102(e.g., made of glass, metal, rubber, etc.), including the bottom of thechannel, the walls, and the bonding surface, thereby creating thecounter electrode in the etched channels and the deposits of metal forfusing in the areas between the channels in one operation. As a result,the manufacturing process is simplified and less costly.

In some embodiments, TiN on a material is utilized to make directconnection(s) from the counter electrode to external electricalpotential, voltage, and/or current sources and/or sinks. For example,for the flow channel component 1902 of Figure in 19, if a top plate orsubstrate included in flow channel component 1902 is made of aconductive material (e.g., metal) that is in conductive contact withcounter electrode 1906, an electrical connector that is at least in partmade of TiN (e.g., TiN coated over a different metal material) isutilized to make a connection between the top plate/substrate of flowchannel component 1902 to a component (e.g., circuit board contact)connected to an external electrical potential, voltage, and/or currentsource and/or sink. If a top plate/substrate of flow channel component1902 is made of a non-conductive material (e.g., glass), a conductivepath (e.g., made at least in part using TiN) is established through oraround the top plate/substrate (e.g., using a via that go through thetop plate and the via is fluid-tight to not allow leakage from the flowchannel path of flow channel component 1902) to counter electrode 1906.Using an electrical connector that is at least in part made of TiN, thisconductive path may be connected to a component (e.g., circuit boardcontact) that is connected to an external electrical potential, voltage,and/or current source and/or sink.

Similarly for the flow channel component 2002 of FIG. 20, if a top plateor substrate included in flow channel component 2002 is made of aconductive material (e.g., metal) that is in conductive contact withcounter electrode 2006, an electrical connector that is at least in partmade of TiN (e.g., TiN coated over a different metal material) isutilized to make a connection between the top plate/substrate of flowchannel component 2002 to a component (e.g., circuit board contact)connected to an external electrical potential, voltage, and/or currentsource and/or sink. If a top plate/substrate of flow channel component2002 is made of a non-conductive material (e.g., glass), a conductivepath (e.g., made at least in part using TiN) is established through oraround the top plate/substrate (e.g., using a via that go through thesubstrate and the via is fluid-tight to not allow leakage from the flowchannel path of flow channel component 2002) to counter electrode 2006.Using an electrical connector that is at least in part made of TiN, thisconductive path may be connected to a component (e.g., circuit boardcontact) that is connected to an external electrical potential, voltage,and/or current source and/or sink.

In some embodiments, TiN material is used (i) as the counter electrode(e.g., counter electrode 1906 in FIG. 19 or counter electrode 2006 inFIG. 20) and/or (ii) as a layer to facilitate bonding to the sequencingchip, and also utilized in making a direct connection from the counterelectrode to external electrical potential, voltage, and/or currentsources and/or sinks.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

What is claimed is:
 1. A nanopore cell, comprising: a titanium nitride(TiN) counter electrode configured to be at a first electric potential;a working electrode configured to be at a second electric potential; andan insulating wall, wherein the insulating wall and the workingelectrode form at least a portion of a well configured to contain anelectrolyte at a voltage that is at least a portion of an electricpotential difference between the first electric potential of thetitanium nitride counter electrode and the second electric potential ofthe working electrode.
 2. The nanopore cell of claim 1, wherein thecounter electrode includes a base material that has been sputtered withtitanium nitride.
 3. The nanopore cell of claim 1, wherein the nanoporecell is a part of a nanopore-based sequencing system and thenanopore-based sequencing system includes a fluid chamber component anda sequencing chip.
 4. The nanopore cell of claim 3, wherein the fluidchamber component forms at least a portion of a winding fluid chamber.5. The nanopore cell of claim 3, wherein the titanium nitride counterelectrode is included on the fluid chamber component.
 6. The nanoporecell of claim 3, wherein titanium nitride is sputtered within a channelsurface of the fluid chamber component.
 7. The nanopore cell of claim 3,wherein titanium nitride is sputtered on an entire surface of the fluidchamber component coupled to the sequencing chip.
 8. The nanopore cellof claim 3, wherein the fluid chamber component and the sequencing chipare coupled together by heating an intermediate metal layer.
 9. Thenanopore cell of claim 3, wherein the sequencing chip includes aplurality of different nanopore sensors and each nanopore sensorincludes a surface for receiving a fluid.
 10. The nanopore cell of claim3, wherein the fluid chamber component and the sequencing chip togetherform at least a portion of a fluid chamber with an inlet delivering afluid into the fluid chamber and an outlet delivering the fluid out ofthe fluid chamber.
 11. The nanopore cell of claim 1, wherein titaniumnitride is utilized in a material utilized to make a connection betweenthe counter electrode and an electrical potential source or sink. 12.The nanopore cell of claim 1, wherein a common electrode is sharedamongst a plurality of different nanopore cells.
 13. The nanopore cellof claim 1, wherein the working electrode is made at least in part oftitanium nitride (TiN).
 14. The nanopore cell of claim 1, wherein theworking electrode comprises a spongy and porous TiN working electrodethat is deposited by a deposition technique with conditions tuned todeposit sparsely-spaced TiN columnar structures or columns of TiNcrystals above a conductive layer.
 15. The nanopore cell of claim 14,wherein the spongy and porous TiN working electrode has a specificsurface area that is ten to a thousand times that of a specific surfacearea of a flat TiN working electrode with substantially identicaldimensions.
 16. The nanopore cell of claim 14, wherein the spongy andporous TiN working electrode has an electrochemical capacitance that isten to a thousand times that of an electrochemical capacitance of a flatTiN working electrode with substantially identical dimensions.
 17. Thenanopore cell of claim 14, wherein the deposition technique comprisesdirect current (DC) reactive sputtering from a titanium target, and theconditions tuned to deposit sparsely-spaced TiN columnar structures orcolumns of TiN crystals above the conductive layer comprise using a lowtemperature, low substrate bias voltage, and high pressure forsputtering.
 18. The nanopore cell of claim 14, wherein a portion of theinsulating wall covers a portion of the working electrode, and whereinthe well has an opening above an uncovered portion of the workingelectrode, and wherein a base surface area of the working electrode isgreater than a base surface area of the opening above the uncoveredportion of the working electrode.
 19. The nanopore cell of claim 18,wherein the base surface area of the working electrode and the basesurface area of the opening above the uncovered portion of the workingelectrode are selected based on a ratio of a capacitance associated withthe working electrode and a capacitance associated with a membrane thatspans across the opening.
 20. The nanopore cell of claim 14, wherein aportion of the insulating wall covers a portion of the workingelectrode, and wherein the well has an opening above an uncoveredportion of the working electrode, and wherein the electrolyte candiffuse through spaces between sparsely-spaced TiN columnar structuresor columns of TiN crystals and diffuse vertically down the uncoveredportion of the working electrode and then horizontally to the coveredportion of the working electrode.